Peptides for modulating t-cell activity and uses therof

ABSTRACT

The present invention provides methods and materials for treating and preventing autoimmune diseases. In particular, the present invention relates to the discovery that small peptides are capable of interacting with CD40, thereby interfering with the ability of CD40 to interact with CD154, which is important in inflammation. The present invention also relate to the use of such peptides in reducing the inflammatory response, and in particular, the autoimmune inflammatory response. The present invention also relates to the use of such short peptides to prevent or reverse autoimmune disease, and particular, diabetes, in individuals suffering from such disease. It also relates to methods and materials for detecting T-cells that express CD40 (Th40 cells). Also provided are kits for reducing inflammation, treating autoimmune diseases, or detecting Th40 cells.

FIELD OF THE INVENTION

The present invention relates to peptides that inhibit the interactionof CD40 and CD154, and the use of such compounds in modulating T-cellactivity and in treating disease.

BACKGROUND

Inflammation normally occurs in response to infection by invadingmicro-organisms. This inflammatory response is beneficial because it isan important part in localizing the infecting agent for removal by theimmune system. However, in autoimmunity there is no infection, yetsevere inflammation is present. The inflammation in this case, referredto as aseptic chronic inflammation, is detrimental since it destroysnormal tissues. The results of this aseptic inflammation arelife-altering and in some cases life-threatening. Moreover, as withacute inflammation, this process is mediated by immune cells, includingT-cells.

A major concern for modern medicine is how to control aseptic, chronicinflammation (ACI) such as that which occurs during autoimmune diseases,as well as how to control acute inflammation resulting from trauma.Inflammation, both chronic and acute, leads to tissue degeneration andeventual loss of function of major organs. ACI is not limited to asingle disease, but is instrumental in numerous autoimmune diseasesincluding, but not limited to type 1 diabetes, multiple sclerosis,systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease,inflammatory bowel disease, chronic obstructive pulmonary diseaseincluding types of autoimmune asthma, atherosclerosis, vasculitis,hypertension, thyroiditis including Hashimoto's and Graves diseases,primary biliary cirrhosis, Paget's disease, Addison's disease, acuterespiratory distress syndrome, acute lung injury, and ACI associatedwith organ transplantation.

Autoimmune disorders are classified into two types: organ-specific(directed mainly at one organ) and non-organ-specific (widely spreadthroughout the body). Examples of organ-specific autoimmune disordersare insulin-dependent Type 1 diabetes which affects the pancreas;Hashimoto's thyroiditis and Graves' disease, which affect the thyroidgland; pernicious anemia, which affects the blood; Addison's disease,which affects the adrenal glands; chronic active hepatitis, whichaffects the live; myasthenia gravis which affects the muscle; andmultiple sclerosis, which affects tissue of the nervous system. Anexample of a non-organ-specific autoimmune disorders is rheumatoidarthritis. Autoimmune diseases are often chronic, debilitating, andlife-threatening. The National Institutes of Health (NIH) estimates thatup to 23.5 million Americans suffer from autoimmune disease and that theprevalence is rising. It has been estimated that autoimmune diseases areamong the ten leading causes of death among women in all age groups upto 65 years.

Acute inflammation, as observed during trauma or sepsis, is also immunecell mediated. While all of the molecular mediators in this process havenot yet been identified, a prominent role for T cells,macrophages/monocytes, neutrophils etc., is strongly implicated.Therefore a means to modulate these cell types would necessarily controlthe inflammatory response.

A unique T cell subset has been shown to be instrumental in thedevelopment of autoimmune disease. These cells are phenotypicallycharacterized as CD4loCD40+ (Waid, D. M., G. M. Vaitaitis, and J.Wagner, D. H. 2004. European Journal of Immunology 34:1488; Vaitaitis,G. M., M. Poulin, R. J. Sanderson, K. J. Haskins, and D. H. Wagner Jr.2003. Cutting Edge, J. Immunol. 170:3455; Wagner, D. H., Jr., G.Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, and K. Haskins. 2002. ProcNatl Acad Sci USA 99:3782; Wagner, D. H., Jr., E. Newell, R. Sanderson,J. H. Freed, and M. K. Newell. 1999. International Journal of MolecularMedicine 4:231), and are referred to as Th40 cells. CD40 expressiontypically is associated with antigen presenting cells and the majorityof prior art describes CD40 as being expressed on B cells, macrophages,monocytes etc. However CD40 proteins are also expressed on T cells(Waid, D. M., G. M. Vaitaitis, and J. Wagner, D. H. 2004. EuropeanJournal of Immunology 34/1488; Vaitaitis, G. M., M. Poulin, R. J.Sanderson, K. J. Haskins, and D. H. Wagner Jr. 2003. Cutting Edge, J.Immunol. 170-3455; Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M.Poulin, C. Dobbs, and K. Haskins. 2002. Proc Natl Acad Sci USA 99:3782;Wagner, D. H., Jr., E. Newell, R. Sanderson, J. H. Freed, and M. K.Newell. 1999. International Journal of Molecular Medicine 4:231;Bourgeois, C., B. Rocha, and C. Tanchot. 2002. Science 297:2060;Fanslow, W. C., K. N. Clifford, M. Seaman, M. R. Alderson, M. K.Spriggs, R. J. Armitage, and F. Ramsdell. 1994. Journal of Immunology152:4262; Ramsdell, F., M. S. Seaman, K. N. Clifford, and W. C. Fanslow.1994. Journal of Immunology 152:2190; Grabstein, K. H., C. R.Maliszewski, K. Shanebeck, T. A. Sato, M. K. Spriggs, W. C. Fanslow, andR. J. Armitage. 1993. Journal of Immunology 150:3141; Armitage, R. J.,C. R. Maliszewski, M. R. Alderson, K. H. Grabstein, M. K. Spriggs, andW. C. Fanslow. 1993. Seminars in Immunology 5:401; Cooper, C. J., G. L.Turk, M. Sun, A. G. Farr, and P. J. Fink. 2004. J Immunol 173:6532).While Th40 cells comprise a proportion of the peripheral CD4+compartment in naïve, non-autoimmune mice (Waid, D. M., G. M. Vaitaitis,and J. Wagner, D. H. 2004. European Journal of Immunology 34:1488;Wagner, D. H., Jr., E. Newell, R. Sanderson, J. H. Freed, and M. K.Newell. 1999. International Journal of Molecular Medicine 4:231), and inhumans (Waid. D. M, R. J. Wagner, A. Putnam, G. M. Vaitaitis, N. D.Pennock, D. C. Calverley, P. Gottlieb, and D. H. Wagner, Jr. 2007. ClinImmunol 124:138), this proportion is drastically expanded to as much as50% of the CD4+ compartment in autoimmune prone mice (Waid, D. M., G. M.Vaitaitis, and J. Wagner. 2004. European Journal of Immunology 34:1488;Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, andK. Haskins. 2002. Proc Natl Acad Sci USA 99:3782; Wagner, D. H., Jr., E.Newell, R. Sanderson, J. H. Freed, and M. K. Newell. 1999. InternationalJournal of Molecular Medicine 4:231) and humans (Waid. D. M, R. J.Wagner, A. Putnam, G. M. Vaitaitis, N. D. Pennock, D. C. Calverley, P.Gottlieb, and D. H. Wagner, Jr. 2007. Clin Immunol 124:138). These Tcells do not express early activation markers and occur in the naïvephenotype of non-challenged mice. In diabetic NOD mice, Th40 cells occurat exaggerated levels in spleen, lymph nodes and the pancreas, evenprior to diabetes onset (Waid, D. M., G. M. Vaitaitis, and J. Wagner, D.H. 2004. European Journal of Immunology 34:1488; Wagner, D. H., Jr., G.Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, and K. Haskins. 2002. ProcNatl Acad Sci USA 99:3782). An elevated number and percentage of these Tcells is seen in peripheral blood of type 1 diabetic patients whencompared to non-autoimmune controls and type 2 diabetic patients (Waid.D. M, R. J. Wagner, A. Putnam, G. M. Vaitaitis, N. D. Pennock, D. C.Calverley, P. Gottlieb, and D. H. Wagner, Jr. 2007. Clin Immunol124:138).

The observed increase in Th40 cells could mean that those T cells areantigen responsive or that CD40 expression is activation induced.Furthermore, several diabetogenic T cell clones are CD40+

(Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs,and K. Haskins. 2002. Proc Natl Acad Sci USA 99:3782). Purified primaryTh40 cells from diabetic NOD mice and from pre-diabetic NOD (12—weeks ofage) mice successfully transfer type 1 diabetes to NOD.scid recipients,directly demonstrating pathogenicity of that T cell subset (Waid, D. M.,G. M. Vaitaitis, and J. Wagner, D. H. 2004. European Journal ofImmunology 34:1488; Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M.Poulin, C. Dobbs, and K. Haskins. 2002. Proc Natl Acad Sci USA 99:3782).It has been shown that Th40 cells infiltrate islet beta cells destroyinginsulin production thus suggesting islet antigen specificity (Waid, D.M., G. M. Vaitaitis, and J. Wagner, D. H. 2004. European Journal ofImmunology 34:1488; Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M.Poulin, C. Dobbs, and K. Haskins. 2002. Proc Natl Acad Sci USA 99:3782).It has also been shown that Th40 cells are required for diabetestransfer. Peripheral (spleen and regional lymph node) T cells that wereCD40 depleted, then CD25, Treg, depleted were not capable oftransferring diabetes to Scid recipients. Even though Tregs wereremoved, if the autoaggressive CD40+ T cells subset is absent, diseasetransfer does not occur.

While Th40 cells are important in the development of autoimmunity,another important factor is expression of the CD40—Ligand, CD154. CD154is temporally induced on activated T cells in response to CD3/TCRstimulation (Lederman, S., M. Yellin, A. Krichevsky, J. Belko, J. Lee,and L. Chess. 1992. Journal of Experimental Medicine 175:1091). CD154expression has also been demonstrated on platelets, monocytes,basophils, eosinophils, dendritic cells, fibroblasts, smooth muscle, andendothelial cells (Russo, S., B. Bussolati, I. Deambrosis, F. Mariano,and G. Camussi, 2003. J Immunol 171:5489; Stumpf, C., C. Lehner, S.Eskafi, D. Raaz, A. Yilmaz, S. Ropers, A. Schmeisser, J. Ludwig, W. G.Daniel, and C. D. Garlichs. 2003. Eur J Heart Fail 5:629; Schonbeck, U.,and P. Libby. 2001. Cell Mol Life Sci 58:4). CD154 is a member of thetumor necrosis factor (TNF) super-family and a soluble form of CD154(sCD154) has been described (Russo, S., B. Bussolati, I. Deambrosis, F.Mariano, and G. Camussi. 2003. J Immunol 171:5489; Stumpf, C., C.Lehner, S. Eskafi, D. Raaz, A. Yilmaz, S. Ropers, A. Schmeisser, J.Ludwig, W. G. Daniel, and C. D. Garlichs. 2003. Eur J Heart Fail 5:629;Toubi, E. and Y. Shoenfeld. 2004 Autoimmunity 37:457). Therefore, sCD154may act like a cytokine (Stumpf, C., C. Lehner, S. Eskafi, D. Raaz, A.Yilmaz, S. Ropers, A. Schmeisser, J. Ludwig, W. G. Daniel, and C. D.Garlichs. 2003. Eur J Heart Fail 5:629). Even though CD154 has not beengenetically linked in T1D studies, sCD154 is significantly elevated inT1D and may play a role in the disease process (Varo, N., D. Vicent, P.Libby, R. Nuzzo, A. L. Calle-Pascual, M. R. Bernal, A. Fernandez-Cruz,A. Veves, P. Jarolim, J. J. Varo, A. Goldfine, E. Horton, and U.Schonbeck. 2003. Circulation 107:2664; Cipollone, F., F. Chiarelli, G.Davi, C. Ferri, G. Desideri, M. Fazia, A. lezzi, F. Santilli, B. Pini,C. Cuccurullo, S. Tumini, A. Del Ponte, A. Santucci, F. Cuccurullo, andA. Mezzetti. 2005. Diabetologia 48:1216; Devaraj, S., N. Graser, S.Griffen, J. Wang-Polagruto, E. Miguelino, and I. Jialal. 2006. Diabetes55:774). The importance of CD40-CD154 interaction in autoimmunity hasbeen established (Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M.Poulin, C. Dobbs, and K. Haskins. 2002. Proc Natl Acad Sci USA 99:3782;Kobata, T., M. Azuma, H. Yagita, and K. Okumura. 2000. Rev. Immunogenet2:74; Homann, D., A. Jahreis, T. Wolfe, A. Hughes, B. Coon, M. J. vanStipdonk, K. R. Prilliman, S. P. Schoenberger, and M. G. von Herrath.2002. Immunity 16:403; Goodnow, C. C. 2001. Lancet 357:2115; Balasa, B.,T. Krahl, G. Patstone, J. Lee, R. Tisch, H. O. McDevitt, and N.Sarvetnick. 1997. Journal of Immunology 159:4620). Blocking CD40-CD154interaction prevents collagen induced arthritis (Durie, F. H., R. A.Fava, T. M. Foy, A. Aruffo, J. A. Ledbetter, and R. J. Noelle. 1993.Science 281:1328) experimental autoimmune encephalitis (Howard, L. M.,and S. D. Miller. 2004. Autoimmunity 37:411), prostatitis (Grossman, M.E., E. Davila, and E. Celis. 2001. J Immunother 24:237), and importantlytype 1 diabetes in the NOD mouse model (Balasa, B., T. Krahl, G.Patstone, J. Lee, R. Tisch, H. O. McDevitt, and N. Sarvetnick. 1997.Journal of Immunology 159:4620). In the diabetes model it was essentialto administer a CD154 blocking antibody to NOD mice at 3-weeks of age;at 9-weeks, blocking antibodies had no effect on diabetes prevention(Balasa, B., T. Krahl, G. Patstone, J. Lee, R. Tisch, H. O. McDevitt,and N. Sarvetnick. 1997. Journal of Immunology 159:4620).

Previous work has also demonstrated that the Th40 cell subset inducesRAG1 and RAG2 transcription, translation and nuclear translocation(Vaitaitis, G. M., M. Poulin, R. J. Sanderson, K. J. Haskins, and D. H.Wagner Jr. 2003. Cutting Edge, J. Immunol. 170:3455) when CD40 isengaged. CD3 engagement does not induce RAG1 or RAG2 in T cells(Vaitaitis, G. M., M. Poulin, R. J. Sanderson, K. J. Haskins, and D. H.Wagner Jr. 2003. Cutting Edge, J. Immunol. 170:3455). Subsequent toRAG1/RAG2 induction, CD40-mediated TCR revision occurs in peripheral Tcells (Vaitaitis, G. M., M. Poulin, R. J. Sanderson, K. J. Haskins, andD. H. Wagner Jr. 2003. Cutting Edge, J. Immunol. 170:3455). CD40induction of TCR revision is RAG dependent. T cells isolated from aTCR-Tg mouse undergo TCR revision when CD40 engaged, but T cells fromthe TCR-Tg.RAG−/− mouse do not TCR revise when CD40 engaged (Wagner, D.H., Jr., E. Newell, R. Sanderson, J. H. Freed, and M. K. Newell. 1999.International Journal of Molecular Medicine 4:231).

Multiple treatment options have been put forward to address and controlboth chronic and acute inflammation. Many approaches use non-steroidalanti-inflammatory drugs (NSAIDS) that attack the production ofleukotrienes and prostaglandins, cellular products that cause localizedinflammation. Other approaches use more powerful immunosuppressant drugssuch as cyclophosphamide, methotrexate and azathioprine that suppressthe immune response and stop the progression of the disease. Still othertreatments involve the use of monoclonal antibodies designed to alterthe immune responses to self-tissues, as occurs during autoimmunediseases. However, all of these treatments often have severe, long-termside effects.

Thus, there exists a need in the art for safer and more effectivemethods for treatment and prevention of autoimmune diseases. The presentinvention addresses this need by describing a novel method for treatmentof autoimmune diseases.

SUMMARY OF THE INVENTION

The present invention provides a novel method for modulatinginflammation, and in particular, inflammation that arises as a result ofan autoimmune disease. The invention is based on the knowledge thatinteraction of CD40-ligand (CD154 protein) with CD40 protein expressedon T-cells (Th40 cells), is important in the development of autoimmunedisease. The invention is also based on the elucidation of the criticalresidues in CD40 and CD154 that are important for this interaction. Thepresent invention relates to blocking the interaction between a CD40protein and a CD154 protein through the use of small peptides thatinteract with the CD40 protein at a site where the CD154 protein wouldnormally bind. The present invention also relates to using such peptidesto reduce the level of Th40 cells, thereby reducing the severity ofdisease. Finally the present invent also relates to novel methods fordetecting Th40 cells.

One embodiment of the present invention is a peptide that interacts witha CD40 protein in such a manner as to modulate inflammation. Preferredpeptides are those that are less than 25 amino acids in length, and thatbind to a CD40 protein, thereby inhibiting its interaction with a CD154protein. Preferred peptides are those that comprise a portion of theCD40 binding site from a CD154 protein.

One embodiment of the present invention is a method to inhibit theinteraction between a CD40 protein and a CD154 protein, the methodcomprising contacting the CD40 protein with a peptide that interactswith the CD40 protein. Preferred peptides interact with the CD40 proteinat the CD154—binding site. Preferably such peptides are less than 20amino acids in length. Even more preferred peptides are those consistingof an amino acid sequence selected from the group consisting of SEQ IDNOs 3-10.

One embodiment of the present invention is a method to modulateinflammation in an animal or a culture of cells, the method comprisingadministering to said animal, or said cells, a peptide that interactswith a CD40 protein in such a manner as to modulate inflammation.Preferred peptides are those that interact with the CD40 protein at theCD154—binding site, thereby modulating inflammation. Preferred peptidesmodulate inflammation by reducing the level of Th40 cells to no morethan 25% of the total T-cell population. Such methods can be used totreat autoimmune diseases such as diabetes.

Another embodiment of the present invention is a means to detectautoagressive T-cells, the method comprising contacting a population ofT-cells with a peptide that binds the CD40 protein, and detecting theCD-40 bound peptide.

One embodiment of the present invention is a method to identify apatient at risk for developing an autoimmune disease, the methodcomprising obtaining a sample containing T-cells from a patient to betested, contacting the sample with a peptide that binds the CD40protein, detecting the CD-40 bound peptide, and determining the level ofTh40 cells from the amount of CD40 bound, wherein a level of Th40 cellsgreater than 25% of the total T-cell population indicates the patient isat risk for developing an autoimmune disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Effect of various peptides of CD154 on the development ofdiabetes in NOD mice

FIG. 2 Effect of a 15-mer peptide from CD154 on the CD4/CD8 ration inNOD mice

FIG. 3 Reversal of diabetes in NOD mice using a 15-mer peptide fromCD154

FIG. 4 Detection of Th40 cells using a 15-mer peptide from CD154

FIG. 5 Screening of B cells using a 15-mer peptide from CD154

FIG. 6 Comparison of Th40 cell levels in diabetic and non-diabetic mice

FIG. 7. Effect of treatment with the 15-mer peptide on insulingranulation of the pancreas

FIG. 8. Effect of mutations in the 15-mer peptide on the ability of the15-mer peptide to inhibit development of diabetes in NOD mice

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that a unique subset ofT-cells, which express CD40 protein, and thus are referred to as Th40cells, is instrumental in autoimmune inflammation. Moreover, involvementof Th40 cells in the autoimmune process is dependent on the interactionbetween CD40 protein expressed on the surface of the T-cell, and CD154protein. Interaction of CD40 and CD154 results in activation signalsbeing delivered between the cells, and subsequent activation of the Th40cell. Such activation results in propagation of the Th40 cell and anincrease in inflammation (e.g., an increase in the number of immunecells and immunoregulatory molecules, present in the system).Accordingly, inhibition of the CD40/CD154 interaction can modulate Th40cell activity, and thereby affect inflammation. Thus the presentinvention relates to peptides that affect the interaction between a CD40protein and a CD154 protein, thereby modulating inflammation. Inparticular, the present invention relates to peptides that affect theinteraction between CD40 protein expressed on the surface of a T-cell,and a CD154 protein, thereby affecting T-cell activity and modulatinginflammation. The invention also relates to methods of using suchpeptides to modulate inflammation and to treat autoimmune disease. Thepresent invention also encompasses the use of such peptides to detectTh40 cells.

Before the present invention is further described, it is to beunderstood that this invention is not strictly limited to particularembodiments described, as such may of course vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It should further be understood thatas used herein, the term “a” entity or “an” entity refers to one or moreof that entity. For example, a nucleic acid molecule refers to one ormore nucleic acid molecules. As such, the terms “a”, “an”, “one or more”and “at least one” can be used interchangeably. Similarly the terms“comprising”, “including” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited. The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed. In addition, all sub-combinations are also specificallyembraced by the present invention and are disclosed herein just as ifeach and every such sub-combination was individually and explicitlydisclosed herein.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

Furthermore, as used herein the term animal refers to a vertebrate,preferably a mammal, more preferably a human. Suitable mammals on whichto use the methods of the present invention include but are not limitedfarm animals, sports animals, pets, primates, mice, rats, horses, dogs,cats, and humans. The term animal can be used interchangeably with theterms subject or patient.

One embodiment of the present invention is a peptide that interacts witha CD40 protein in such a manner as to modulate inflammation. As usedherein, the terms interact, interaction, and the like, mean that twomolecules come into sufficient physical proximity such that they cause amodulation of inflammation. One type of interaction is a bindinginteraction. In such an interaction the peptide associates with CD40 toform a complex. An example of complex formation is the association of anantigen with an antibody. According to the present invention, binding ofa peptide of the present invention to a CD40 protein can be reversible(e.g., non-covalent binding interactions) or non-reversible (e.g.,covalent binding interactions). Moreover, a reversible interaction canbe strong or weak, the strength of the interaction being determined bythe forces (e.g., ionic charges, hydrogen binding, van der Wallsinteractions, etc.) exerted by each protein on the other protein in thecomplex. Factors affecting the strength of an interaction between twomolecules are known to those skilled in the art. One useful measure ofthe strength of binding between two molecules, such as a peptide and aprotein, is the dissociation constant (Kd). Preferred peptides of thepresent invention are those that bind to a CD40 protein with a Kd of nomore than about 1×10⁻⁶ M, about 1×10⁻⁷ M, or about 1×10⁻⁸M. Particularlypreferred peptides are those having a Kd of less than about 1×10⁻⁹M. Inone embodiment, a peptide of the present invention binds to a CD40protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM,less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or lessthan 1 nM. Methods of measuring and analyzing binding interactionsbetween a peptide and a CD40 protein are known by those of skill in theart.

As used herein, to modulate inflammation means to change the level ofTh40 cells present in an animal, or in a culture of T-cells. As usedherein, the terms level, number, count and concentration can be usedinterchangeably. Modulation of inflammation can mean an increase ordecrease in the number of Th40 cells present in the inflammatoryenvironment. Consequently, modulation can be referred to as positive ornegative. Positive modulation (also referred to as up-regulation) ofinflammation refers to an increase in the number of Th40 cells in theinflammatory environment. Negative modulation (also referred to asdown-regulation) of inflammation refers to a reduction in the number ofTh40 cells present in the inflammatory environment. A preferred peptideis one that down-regulates inflammation, thereby reducing the number ofTh40 cells present in the inflammatory environment. Positive andnegative modulation of inflammation may or may not result in a change inthe type and amount of immunoregulatory molecules present in theinflammatory environment.

It will be appreciated by those skilled in the art that both a cellculture system and the immune system of an animal comprise basal levelsof immune cells and immunoregulatory molecules. The phrases basal leveland normal level can be used interchangeably. With regard to the immunesystem of an animal, as used herein, the basal level of a type of immunecell (e.g., Th40 cell), or a immunoregulatory molecule, refers to theaverage number of that cell type, or immunoregulatory molecule, presentin a population of individuals considered healthy (i.e., free ofmetabolic, autoimmune, or infectious disease). With regard to a cellculture system, as used herein, the basal level of a type of immunecell, or an immunoregulatory molecule, refers to the average level ofthat cell type, or immunoregulatory molecule, present in a population ofcells that is non-activated. Those skilled in the art are capable ofdetermining if a T-cell, or a population of such cells, is activated.For example, the expression of CD69, CD25 and/or CD154 proteins by acell indicates that the cell has been activated.

The basal level of a cell or molecule can be a specific amount (e.g., aspecific concentration) or it can encompass a range of amounts. Basallevels, or ranges, of immune cells and immunoregulatory molecules areknown to those in the art. For example, in a healthy individual, thenormal level of CD4+ T-cells present in human blood is 500-1500cells/ml. Variability in this measurement can result from differences inthe method used to determine the cell count. Furthermore, normal levelsof cells can also be reported as a percentage of a total cellpopulation. For example, in a healthy individual, Th40 cells make upless than 25% of the total T cell population. Thus, as used herein, theterm inflammation refers to an inflammatory environment in which Th40cells make up greater than about 25%, greater than about 30%, greaterthan about 35%, greater than about 40%, greater than about 45%, greaterthan about 50%, greater than about 55%, greater than about 60%, greaterthan about 65%, greater than about 70%, greater than about 75%, orgreater than about 80% of the total T-cell population. Moreover, apreferred peptide of the present invention is one that reduces the levelof Th40 cells to less than about 50%, less than about 45%, less thanabout 40%, less than about 35%, less than about 30%, less than about27%, or equal to about 25% of the total T-cell population. Methods ofmeasuring different types of T-cells in the T-cell population are knownto those skilled in the art. Furthermore, a novel method for detectingTh40 cells using peptides of the present invention is disclosed herein.

As used herein, the phrase inflammatory environment refers to theoverall population of immune cells, and related immunoregulatorymolecules, that are present in a culture of cells, or in the body of ananimal. As such, the phrase inflammatory environment encompasses thetypes, and/or the relative amounts of immune cells and immunoregulatorymolecules (e.g., cytokines) present in a culture of cells, or in ananimal, which are involved in affecting an inflammatory reaction.Examples of cells encompassed by the term inflammatory environmentinclude, but are not limited to, T cells, neutrophils, macrophages,granulocytes, and the like. The inflammatory environment relates tocells and molecules that mediate both acute and chronic inflammation. Itwill be appreciated by those skilled in the art that the inflammatoryenvironment refers to the system to which peptides of the presentinvention are administered. In one embodiment, the system is a cellculture system. In one embodiment, the system is a whole animal.

A preferred peptide of the present invention is one that selectivelyinteracts with a CD40 protein in solution, as determined using an assaysuch as an immunosorbent assay, or on the surface of a T-cell. As usedherein, the terms selectively, selective, specific, and the like,indicate the peptide has a greater affinity for a CD40 protein than itdoes for proteins unrelated to the CD40 protein. More specifically, theterms selectively, selective, specific, and the like indicate that theaffinity of the peptide for CD40 is statistically significantly higherthan its affinity for a negative control (e.g., an unrelated proteinsuch as albumin) as measured using a standard assay (e.g., ELISA).Suitable techniques for assaying the ability of a peptide to selectivelyinteract with a CD40 protein are known to those skilled in the art. Suchassays can be in vitro or in vivo assays. Examples of useful assaysinclude, but are not limited to, an enzyme-linked immunoassay, acompetitive enzyme-linked immunoassay, a radioimmunoassay, afluorescence immunoassay, a chemiluminescent assay, a lateral flowassay, a flow-through assay, an agglutination assay, a particulate-basedassay (e.g., using particulates such as, but not limited to, magneticparticles or plastic polymers, such as latex or polystyrene beads), animmunoprecipitation assay, an immunoblot assay (e.g., a western blot), aphosphorescence assay, a flow-through assay, a chromatography assay, apolyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmonresonance assay, a spectrophotometric assay, a particulate-based assay,an electronic sensory assay and a flow cytometric assay. Methods ofperforming such assays are well known to those skilled in the art. Inone embodiment, an assay can be performed using cells in culture, or itcan be performed in a whole animal. Assays can be designed to givequalitative, quantitative or semi-quantitative results, depending on howthey are used and the type of result that is desired.

One embodiment of the present invention is a peptide that interacts witha CD40 protein in such a manner as to affect the interaction of the CD40protein with a CD154 protein, thereby modulating inflammation. Theeffect of the peptide on the CD40/CD154 interaction can be positive orit can be negative. For example, the peptide can interact with the CD40protein in such a manner that the strength of the interaction betweenthe CD40 protein and a CD154 protein is increased. Alternatively, thepeptide can interact with the CD40 protein such that the strength of theinteraction between the CD40 protein and a CD154 protein is decreased.Methods of measuring the strength of binding between the peptide and aCD40 protein are known to those skilled in the art. A preferred peptideof the present invention is one that reduces the strength of theinteraction between a CD40 protein and a CD154 protein. Preferredpeptides of the present invention reduce the strength of binding betweena CD40 protein and a CD154 protein by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%. A particularly preferredpeptide is one that completely inhibits binding of CD40 to CD154.Complete inhibition of binding between CD40 and CD154 means that when apeptide of the present invention is brought into proximity with a CD40protein and a CD154 protein under conditions that would normally allowthe interaction of CD40 and CD154, no such interaction occurs andactivation signals are not stimulated in the CD40-expressing cell.Consequently CD40/CD154 mediated modulation of inflammation does notoccur. In one embodiment, the peptide interacts with the CD40 protein insuch a manner as to reduce the level of inflammation in the system. Inone embodiment, the peptide interacts with the CD40 protein in such amanner as to inhibit the development of inflammation in the system.

While peptides of the present invention can interact with any site onthe CD40 protein, preferred peptides of the present invention interactwith the CD40 protein at a location that overlaps with the CD154 bindingsite. In one embodiment, a peptide of the present invention interactswith the CD40 protein at the CD514 binding site. An example of such apeptide is a CD40 ligand competitive antagonist. As used herein,peptides that interfere with, or inhibit, the binding of a CD154 proteinto a CD40 protein are referred to as small interfering peptides (SIPs).As used herein a small interfering peptide is a peptide that, throughphysio-chemical properties, interferes with the interaction of a CD40protein with a CD154 protein, thereby preventing activation signals frombeing delivered to the CD40-bearing cell, thus limiting the activationof the CD40-bearing cell, and consequently, inflammation. Asdemonstrated herein, the consequences of such interference areprevention of T-cell activation and propagation, and a prevention orreduction of inflammation.

A peptide useful for practicing methods of the present invention shouldbe of a size sufficient to interact with CD40 protein in such a manneras to modulate inflammation. It is understood by those skilled in theart that preferred peptides are relatively short since they are easierand less expensive to produce. Preferred peptides are those that areless than 20 amino acids in length. A preferred peptide is one that is6, 13 or 15 amino acids in length. In one embodiment, the peptideconsists of an amino acid selected from the group consisting of SEQ IDNO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. The sequences of suchpeptides are shown below in Table 1.

TABLE 1 SEQ ID NO SEQUENCE Description  1MIETYSQPSP RSVATGLPAS MKIFMYLLTV SwissProFLITQMIGSV LFAVYLHRRL DKVEEEVNLH 27548.2EDFVFIKKLK RCNKGEGSLS LLNCEEMRRQ Mouse CD40FEDLVKDITL NKEEKKENSF EMQRGDEDPQ Ligand IAAHVVSEAN SNAASVLQWA KKGYYTMKSN(CD154 LVMLENGKQL TVKREGLYYV YTQVTFCSNR Protein)EPSSQRPFIV GLWLKPSSGS ERILLKAANT HSSSQLCEQQ SVHLGGVFEL QAGASVFVNVTEASQVIHRV GFSSFGLLKL  2 MIETYNQTSP RSAATGLPIS MKIFMYLLTV SwissProFLITQMIGSA LFAVYLHRRL DKIEDERNLH 29965 EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQHuman CD40 FEGFVKDIML NKEETKKENS FEMQKGDQNP LigandQIAAHVISEA SSKTTSVLQW AEKGYYTMSN (CD154 NLVTLENGKQ LTVKRQGLYY IYAQVTFCSNProtein) REASSQAPFI ASLCLKSPGR FERILLRAANTHSSAKPCGQ QSIHLGGVFE LQPGASVFVN VTDPSQVSHG TGFTSFGLLK L  3 KGYYCore-sequence  4 KKGYYT 6-mer  5 AKKGYYTM 8-mer-mouse  6 AEKGYYTM8-mer human  7 VLQWAKKGYYTMKSK 15-mer-mouse  8 VLQWAEKGYYTMSNN15-mer human  9 NAASVLQWAKKGYYTMKSNLVMLE 24-mer 10 ISQAVHAAHAEINEAGR15-mer from ovalbumin; control peptide 11 G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-NGly-1 12 V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2 13V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 14 V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-NGly-4 15 V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-5 16V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-6 17 V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-NGly-7 18 V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-8 19V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-9 20 V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-NGly-10 21 V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N Gly-11 22 ISQAVHAAHAEINEAGR15-mer from ovalbumin; control peptide 23 YVQGKANLKSKLMYT Scrambledpeptide

Interaction of a CD40 protein and a CD154 protein has been shown tooccur at particular regions within each protein. The inventors have nowshown that, surprisingly, a peptide comprising only a short portion ofthe CD154 region that interacts with CD40 is capable of binding to aCD40 protein, thereby modulating inflammation. Thus one embodiment ofthe present invention is a peptide that comprises at least a portion ofthe amino acid sequence of a CD154 protein such that the peptideinteracts with CD40 protein in such a manner as to modulateinflammation. In one embodiment, interaction of the peptide with CD40protein results in negative modulation of inflammation. In one aspect,the peptide comprises at least a portion of SEQ ID NO:1 or SEQ ID NO:2.In a preferred aspect, the peptide is as short as possible yet comprisesenough of the CD154 protein to allow interaction with a CD 40 protein insuch a manner as to modulate inflammation. In one embodiment, a peptideof the present invention comprises 6, 13 or 15 contiguous amino acidsfrom SEQ ID NO:1 or SEQ ID NO:2, and interacts with CD40 in such amanner as to modulate inflammation. A preferred peptide comprises a coresequence consisting of lysine-glycine-tyrosine-tyrosine (KGYY; SEQ IDNO:3), which corresponds to amino acids 142-145 of SEQ ID NO:1 and aminoacids 143-146 of SEQ ID NO:2. Useful peptides can comprise additionalregions of sequence from SEQ ID NO:1 or SEQ ID NO:2 that are adjacent tothe core sequence, so long as the peptide is capable of modulatinginflammation. In one embodiment of the present invention, a peptidecomprises at least one sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, solong as the peptide interacts with CD40 protein in such a manner as tomodulate inflammation. In one embodiment of the present invention, apeptide consists of a sequence selected from the group consisting of SEQID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

While peptides of the present invention can consist entirely ofsequences that are responsible for the interaction of the peptide with aCD40 protein, they may additionally contain amino acid sequences that donot interact with a CD40 protein, but which have other useful functions.Any useful, additional amino acid sequence can be added to theCD40-interacting sequence, so long as the additional sequences do nothave an unwanted affect on the ability of the CD40 interacting sequenceto interact with a CD40 protein. For example, in addition to the aminoacid sequence responsible for interacting with a CD40 protein, a peptideof the present invention can contain amino acid sequences that areuseful for visualizing or purifying the peptide. Such sequences act aslabels (e.g., enzymes) or tags (antibody binding sites). Examples ofsuch labels and tags include, but are not limited to, B-galactosidase,luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotintags, and fluorescent tags. Other useful sequences for labeling andtagging proteins are known to those of skill in the art.

Likewise, peptides of the present invention can be modified, so long assuch modification does not significantly affect the ability of thepeptide to modulate inflammation. Such modifications can be made, forexample, to increase the stability, solubility or absorbability of theprotein. Examples of such modifications include, but are not limited topegylation, glycosylation and chemical modification of the peptide.

Peptides of the instant invention can be obtained from nature (e.g.,obtained from plants, animals or microorganisms) or they can be producedin a laboratory (e.g., recombinantly or synthetically). Preferredpeptides are those that are synthesized. Also encompassed are peptidesthat are combinations of natural and synthetic molecules. Generalmethods for producing and isolating recombinant or synthetic peptidesare known to those skilled in the art. It should be noted that, as usedherein, an isolated, or biologically pure, molecule, is one that hasbeen removed from its natural milieu. As such the terms isolated,biologically pure, and the like, do not necessarily reflect the extentto which the protein has been purified.

As has been described herein, interaction of the CD40 protein and theCD154 protein are necessary for involvement of Th40 cells in autoimmuneinflammation. Consequently, inhibition of the interaction between a CD40and CD154 protein using peptides of the present invention is a usefulmethod of affecting autoimmune inflammation. Thus one embodiment of thepresent invention is a method to reduce the interaction between a CD40protein and a CD154 protein comprising introducing into an environmentcontaining a CD40 protein and a CD154 protein a peptide that interactswith the CD40 protein in such a manner as to reduce the interactionbetween the CD40 protein and the CD154 protein. In one aspect of theinvention, the peptide reduces the interaction between the CD40 proteinand the CD154 protein by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%. In one embodiment, the peptidereduces the interaction between the CD40 protein and the CD154 proteinby a factor of at least 10, at least 100, at least 1,000, at least10,000. Methods of measuring the strength of the interaction between theCD40 protein and the CD154 protein have been discussed previously, andare also know to those of skill in the art.

One embodiment of the present invention is a method to modulateinflammation comprising contacting a CD40 protein with a peptide thatinteracts to the CD40 protein in such a manner as to modulateinflammation. In one aspect of the invention, interaction of the peptidewith the CD40 protein increases the number of Th40 cells by at least 5%,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%. Inone embodiment, interaction of the peptide with the CD40 proteinincreases the number of Th40 cells by a factor of at least 10, at least100, at least 1,000, at least 10,000. One aspect of the presentinvention is a method to reduce inflammation in a patient, the methodcomprising administering a peptide of the present invention to thepatient. In one embodiment, the peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:8, SEQ ID NO:9 and SEQ ID NO:10. In one embodiment, the peptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. In a preferredembodiment, interaction of the peptide with the CD40 protein decreasesthe number of Th40 cells by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%. In another embodiment,interaction of the peptide with the CD40 protein decreases the number ofTh40 cells by a factor of at least 10, at least 100, at least 1,000, atleast 10,000. In a preferred embodiment, the level of Th40 cells isreduced so that Th40 cells comprise no more than about 20%, about 25%,about 30%, about 35%, or about 40% of the total T-cell population.

Peptides and methods of the present invention are suitable for use incell culture as well as for treating a patient. As used herein the termpatient refers to any animal in need of such treatment. The animal canbe a human or a non-human animal. A preferred animal to treat is amammal. A peptide can be administered or applied per se, or aspharmaceutical compositions. A peptide of the present invention, or apharmaceutical composition thereof, can be administered to a patient bya variety of routes, including, but limited to, by injection (e.g.,intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal),by inhalation, by oral (e.g., in a pill, tablet, capsule, powder, syrup,solution, suspension, thin film, dispersion or emulsion.), transdermal,transmucosal, pulmonary, buccal, intranasal, sublingual, intracerebral,intravaginal rectal or topical administration or by any other convenientmethod known to those of skill in the art.

The amount of a peptide of the present invention and/or a pharmaceuticalcomposition thereof that will be effective can be determined by standardclinical techniques known in the art. Such an amount is dependent on,among other factors, the patient being treated, including, but notlimited to the weight, age, and condition of the patient, the intendedeffect of the compound, the manner of administration and the judgment ofthe prescribing physician.

A peptide of the present invention, or a pharmaceutical compositionthereof, can be administered alone or in combination with one or moreother pharmaceutical agents, including other compounds of the presentinvention. The specific pharmaceutical composition depends on thedesired mode of administration, as is well known to the skilled artisan.

Because the inventors have discovered that Th40 cells are intimatelyinvolved in the development of autoimmune diseases, the peptides andmethods disclosed herein can be used to affect inflammation resultingfrom such diseases. Thus, one embodiment of the present invention is amethod to treat autoimmune disease in a patient in need of suchtreatment, the method comprising administering to a patient a peptidethat interacts with the CD40 protein, thereby reducing inflammation. Inone embodiment the peptide interacts with the CD40 protein in such amanner as to affect the interaction of CD40 and CD154, thereby reducinginflammation. In a preferred embodiment, interaction of the peptide withthe CD40 protein reduces the number of Th40 cells in a patient to alevel equal to that observed in subjects that do not have autoimmunedisease. The present invention is suitable for treating any patienthaving an autoimmune disease the development of which is dependent onTh40 cells. More specifically, peptides of the present invention aresuitable for reducing the level of Th40 cells in such patients. In apreferred embodiment, a peptide of the present invention reduces thelevel of Th40 cells in a patient suffering from an autoimmune disease tono more than about 25% of the total T-cell population. Examples of suchdisease included, but are not limited to, asthma, type 1 diabetes;multiple sclerosis; systemic lupus erythematosa; rheumatoid arthritis;Crohn's disease; inflammatory bowel disease; chronic obstructivepulmonary disease (COPD) including types of autoimmune asthma;atherosclerosis; vasculitis; hypertension; thyroiditis includingHashimoto's and Graves diseases; primary biliary cirrhosis; Paget'sdisease; Addison's disease; acute respiratory distress syndrome, acutelung injury; ACI associated with organ transplantation; hypertension,etc.

One example of a disease that is particularly amenable to treatmentusing a peptide of the present invention is diabetes. In diabetes, thebody's production of, or response to, insulin is impaired. Consequentlycells are unable to utilize glucose in the blood, and the levels of thissugar become elevated. Mice are considered diabetic when their bloodglucose level is greater than 250 mg/dl for three consecutive days. Inhumans, a normal, average blood glucose level is 60-110 mg/dl. Howeverdiabetics have blood glucose levels of at least 130 mg/dl, and usuallymuch higher. Thus, one embodiment of the present invention is a methodto prevent diabetes in an individual at risk for developing diabetes,the method comprising administering to the individual a peptide of thepresent invention. Such risk can result from familial factors (e.g.,inheritance) or from other factors, such as the physical condition ofthe individual. Methods of risk assessment are known to those skilled inthe art. In one embodiment, the peptide is administered at a time whenthe individual's blood glucose level is from about 60 mg/dl to about 110mg/dl. In one embodiment, the peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:8, SEQ ID NO:9 and SEQ ID NO:10, so long as the peptide candown-regulate inflammation. In one embodiment, the peptide consists ofan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

The inventors have also shown that, surprisingly, peptide of the presentinvention can be used to reverse the disease process in individualsalready showing signs of diabetes. Thus, one aspect of the presentinvention a method to reverse diabetes comprising administering to apatient diagnosed as having diabetes, a peptide of the presentinvention. In one embodiment, the peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, so long as the peptide candown-regulate inflammation. In one embodiment, the peptide consists ofan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. As usedherein the phrase to reverse diabetes means to reduce the blood glucoselevel of a diabetic individual to a level comparable to that observed ina non-diabetic individual. As noted above, the blood glucose level of anon-diabetic subject is from about 60 mg/dl to about 110 mg/dl. Thus oneaspect of the present invention is a method to reduce the blood glucoselevel in a patient diagnosed as having diabetes to less than 110 mg/dl,and preferably between 60 mg/dl and 110 mg/dl.

As has been described, peptides of the present invention selectivelybind to a CD40 expressing cell. Consequently, peptides of the presentinvention can be used to identify Th40 cells. Thus one embodiment of thepresent invention is a method to detect Th40, said method comprisingcontacting a T-cell population with a peptide of the present invention.In a preferred embodiment, the peptide is labeled with a detectablemarker, such as, for example, luciferase or alkaline phosphatase. Suchdetection can be performed using assay techniques known to those skilledin the art. In general, an assay for detecting Th40 cells using apeptide of the present invention comprises (a) obtaining a sample ofcells; (b) contacting a peptide of the present invention with said cellsunder condition suitable to allow binding of the peptide to Th40 cells,if present; (c) washing said cells using conditions that disruptnon-specific interactions, and that remove unbound peptide; and (d)detecting peptide bound to cells. Detection of bound peptide can beachieved directly or indirectly. For example, direct detection can beachieved using a peptide labeled using a detectable marker, as disclosedherein. Following the wash step listed above, the cells are then simplyscreened for the presence of detectable marker. The presence ofdetectable marker in the cell sample indicates the presence of Th40cells. Alternatively, indirect detection involves the use of a secondmolecule, such as an antibody, that binds to the peptide. In an indirectdetection assay, following the wash step listed above, a detectionmolecule that binds the peptide is added to the cell sample. Thedetection molecule is labeled with a detectable marker. After washingaway unbound detection molecule, the cells are screened for the presenceof detectable marker. The presence of detectable marker in the cellsample indicates the presence of Th40 cells. It should be understoodthat the assays described herein are meant as examples of useful assays,and other assay techniques can be employed. Suitable assay techniquesare known to those skilled in the art, and are also disclosed in, forexample, Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch,E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition(December 1989). All referenced cited herein are incorporated herein intheir entirety.

The assay technology described above can also be used to identify othermolecules that affect the interaction of a CD40 protein with a CD514protein. Examples of such molecules include, but are not limited to,proteins, peptides and small molecules. For example, assays can bedesigned that test the ability of molecules to compete with a peptide ofthe present invention for binding to a Th40 cell. For instance, apeptide labeled with a detectable marker, can be mixed with a testmolecule and a population of cells known to contain Th40 cells, underconditions that allow binding of the peptide to the Th40 cells.Following an appropriate incubation period, the cells are washed toremove unbound peptide, and the cells screened for the presence ofdetectable marker. Alternatively, the labeled peptide could be bound toTh40 cells first, and after a wash step to remove unbound peptide, thetest molecule could be added to the cells containing bound peptide.Following an incubating period and a wash step to remove unboundmolecule, or released peptide, the cells are screened for the presenceof detectable marker. In either case, absence of the detectable markerin the cell sample indicates the test molecule is able to compete withthe peptide for binding to the Th40 cells, while presence of thedetectable marker would indicate the test molecule does not inhibitbinding of the peptide to Th40 cells. Inhibition of binding need not be100%, as such assay would also be useful for identifying molecules thatpartially inhibit binding of the peptide to Th40 cells. It is understoodby those skilled in the art that such assays would involve the use ofpositive controls (e.g., unlabeled peptide) and negative controls (e.g.,a protein/molecule that is known not to bind to Th40 cells).

Because increased levels of Th40 cells are associated with thedevelopment of autoimmune disease, the present invention can be used toidentify patients at risk for developing autoimmune disease. Thus oneembodiment of the present invention is a method to identify a patient atrisk for developing an autoimmune disease. In one embodiment, patientsat risk for developing an autoimmune disease are identified by obtaininga sample from a patient to be tested, contacting the T-cell portion saidsample with a peptide of the present invention, and determining thelevel of Th40 cells present in the sample, wherein a level of Th40 cellsgreater than about 25% of the total T-cell population indicates thepatient is at risk for developing an autoimmune disease. In oneembodiment, the peptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ IDNO:9 and SEQ ID NO:10, so long as the peptide binds to the CD40 protein.In one embodiment, the peptide consists of an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:8, SEQ ID NO:9 and SEQ ID NO:10. In a preferred embodiment thepeptide is labeled with a suitable detectable marker such as, forexample, luciferase or alkaline phosphatase.

The present invention also comprises kits useful for practicing themethods disclose herein. One embodiment is a kit for modulatinginflammation in an animal or in cells in culture, the kit comprising apeptide that interacts with a CD40 protein in such a manner as tomodulate inflammation. In one embodiment, the peptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, so long as the peptidecan down-regulate inflammation. In one embodiment, the peptide consistsof an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. Anotherembodiment is a kit for determining the level of Th40 cells, the kitcomprising a peptide that interacts with a CD40 protein, and means fordetecting CD40-bound peptide. Kits can also contain associated reagentsand components, such as, but not limited to, buffers, labels,containers, inserts, tubing, vials, syringes, and the like.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

This Example demonstrates the effect of various peptide fragments ofCD154 on CD4/CD8 ratios and the development of diabetes in NOD mice.

Peptides were designed based on the amino acid sequence of mouse CD154protein (SEQ ID NO:1) in the SwissPro database. The peptides were thenordered from New England Peptide. The lyophilized peptides weresuspended in sterile saline at 1 mg/ml. 100 ug of a particular peptidewas then injected into the tail vein of 8-9 week old NOD mice. Controlmice received 100 ul of sterile saline. This is well before the onset ofdiabetes, but after damage to pancreatic islets has begun. Three daysafter the initial injection, another 100 ug of peptide (or 100 ul ofsaline in the case of the Control mice) was injected into the tail vein.Mice were then injected with peptide (or saline) on a weekly basis. At10 weeks of age, mice were monitored for diabetes, as indicated by ablood glucose level greater than 250 mg/dL for three consecutive days.The results of this study are shown in FIG. 1. During this time, bloodwas also taken from the tail vein, or by sub-mandibular venal puncture,and the level of CD4+ and CD8+ cells determined by flow cytometry usingantibodies for CD4 protein and CD8 protein. The results of this analysisare shown in FIG. 2.

The results demonstrate that treatment with a peptide unrelated to theCD154 protein did not reduce the development of diabetes in NOD mice. Incontrast, treatment of mice with a 15-mer peptide derived from the CD154protein prevented the onset of diabetes. Further, both the 6-mer and10-mer peptides derived from the CD154 protein had significant effectson the development of diabetes. In addition, the data demonstrate thatthe 15-mer peptide did not result in compromise of the immune system, asdetermined by the CD4/CD8 ratio.

Example 2

This Example demonstrates the effect of the 15-mer peptide onhyperglycemia in newly diabetic NOD mice.

Six mice from that had received the 6-mer peptide in Example 1, and thathad subsequently developed diabetes, were injected intravenously with100 ug of the 15-mer peptide. These mice were then given weeklyinjections of the 15-mer peptide into their tail veins, and their bloodglucose levels monitored twice-weekly. The 15-mer peptide wasadministered for a total of ten weeks, after which the treatment wasstopped. The results of this study are shown in FIG. 3.

This study demonstrates that injection of the 15-mer peptide intoalready diabetic mice can reverse hyperglycemia. It also demonstratesthat cessation of the treatment results in return of hyperglycemiawithin 7 weeks.

Example 3

This study demonstrates the ability of the 15-mer peptide to bind toTh40 cell and B cells.

Total lymphocytes were isolated from 9 week old NOD mice. Thelymphocytes were incubated with anti-CD, anti-CD8, and an FITC-labeled15-mer peptide, and then analyzed by flow cytometry. Cells were gatedfor CD4 (both CD4hi and CD4lo populations were included) and CD4 versusthe 15-mer peptide. The results of this analysis are shown in FIG. 4.

B cells were isolated from the spleens of NOD mice. Sorted MHC-II+ cellswere purified from total lymphocytes. Cells were stained withFITC-labeled 15 mer peptide, anti-CD40, and B cell markers CD19 andCD21. MHC-II+ cells were gated for CD19+ and CD21+ and then 15-merpeptide versus Cd40 antibody was measured. The results of this study areshown in FIG. 5.

This study shows that a substantial majority, 90% of CD40+ T-cells, alsobind the 15-mer peptide, thereby demonstrating that the 15-mer peptideis highly specific for CD40+ cells. It also shows that while 90% of Bcells were CD40 positive, only 8% of B cells bound the 15-mer peptide.

Example 4

This example demonstrates the level of CD40 positive cells in the bloodof type-I diabetic subjects and non-diabetic (control) subjects.

1 ml of whole blood was obtained from each individual and incubated withbiotin-conjugated, 15-mer peptide. The cells were then exposed to horseradish peroxidase (HRP)-avidin, washed and the absorbance at 405 nmdetermined using a spectrophotometer. The results of this study areshown in FIG. 6.

This study demonstrates that blood cells from patients having type-Idiabetes had higher 15-mer peptide binding activity than cells fromnon-diabetic controls.

Example 5

This example demonstrates the level of insulin granulation observed inthe pancreas of NOD mice treated with either the 15-mer peptide or apeptide from ovalbumin.

At the onset of diabetes, six NOD mice were injected with 100 ug/ml ofthe 15-mer peptide (SEQ ID NO:9), resulting in the reversal ofhyperglycemia in 80% of the recipients. Six weeks after reversal ofhyperglycemia, mice were sacrificed and the pancreas removed foranalysis. The pancreas was fixed, sectioned and then stained using analdehyde/fuschsin stain that allows detection of insulin granules.Granulation of the tissue was scored as follows: 4=completelygranulated; 3=75% of islet granulated; 2=50% of islet granulated, andperi-insulitis; 1=25% of islet granulated; 0=no insulin granulesdetected. The results of this analysis are shown in FIG. 7.

This analysis demonstrates that the 15-mer peptide preserved insulingranules in the majority of the mice, and was significantly improved inpeptide-reversed diabetic mice compared to diabetic mice that receivedan irrelevant peptide.

Example 6

This example demonstrates the effect of mutations in the 15-mer peptideon its ability to prevent the onset of diabetes.

Peptide were designed and produced as described in Example 1. Variantpeptides were produced so that in each variant, a glycine wassubstituted for an amino acid corresponding to an amino acid inpositions 1-9 of SEQ ID NO:9, as follows:

Gly-1 G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N (SEQ ID NO: 11) Gly-2V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N (SEQ ID NO: 12) Gly-3V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N (SEQ ID NO: 13) Gly-4V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N (SEQ ID NO: 14) Gly-5V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N (SEQ ID NO: 15) Gly-6V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N (SEQ ID NO: 16) Gly-7V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-N (SEQ ID NO: 17) Gly-9V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N (SEQ ID NO: 18) Gly-10V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N (SEQ ID NO: 19) Gly-11V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-N (SEQ ID NO: 20) Gly-12V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N (SEQ ID NO: 21)

NOD mice were placed in groups of 10, and the mice in each groupinjected IV weekly with 50 ug of either wild-type (WT; Legend) peptideor a variant peptide (in PBD, ph 7.2) listed above. The development ofdiabetes was monitored by measuring blood glucose levels on a weeklybasis. Mice were considered “diabetic” when blood glucose was 250 mg/dlor greater for 2 consecutive readings. Injections began at 6 weeks ofage=pre-diabetes.

This example demonstrates that substitution of a glycine at any ofpositions 1-7, or 9-12, reduces the ability of the 15-mer peptide toinhibit the development of diabetes. It also shows that such mutationsdo not completely abolish the ability of the mutated 15-mer peptide toinhibit the development of diabetes.

What is claimed is:
 1. A peptide that affects the interaction of CD40with CD154/gp39/CD40-ligand in such a manner as to modulateinflammation.
 2. The peptide of claim 1, wherein said peptide binds toCD40.
 3. The peptide of claim 1, wherein said peptide binds to a CD40protein with a Kd of greater than 10⁶.
 4. The peptide of claim 1,wherein said peptide affects the interaction of CD40 and CD154.
 5. Thepeptide of claim 1, wherein said peptide inhibits the binding of CD40 toCD154.
 6. The peptide of claim 1, wherein said peptide binds CD40 at thesite where CD40 interacts with CD154.
 7. The peptide of claim 1, whereinsaid peptide affects the interaction of CD40 with CD154 in such a manneras to prevent the expansion of Th40 cells.
 8. The peptide of claim 1,wherein said peptide affects the interaction of CD40 with CD154 in sucha manner as to reduce the number of Th40 cells.
 9. The peptide of claim1, wherein said peptide affects the interaction of CD40 with CD154 insuch a manner as alter the cytokine expression profile of a cellpopulation treated with said peptide.
 10. The peptide of claim 1,wherein said peptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9and SEQ ID NO:10.
 11. The peptide of claim 1, wherein said peptideconsists an amino acid sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
 12. The peptideof claim 1, wherein said peptide consists an amino acid sequenceselected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10. 13.The peptide of claim 1, wherein said peptide is obtained from naturalsources or it is synthesized.
 14. A method to inhibit the interactionbetween a CD40 protein and a CD154 protein comprising contacting saidCD40 protein with a peptide that binds said CD40 protein at theCD154-binding site, thereby inhibiting the interaction of said CD40 andCD154 proteins. 15-18. (canceled)
 19. A method of reducing the number ofTh40 cells in a patient by administering a compound that inhibits theinteraction of CD40 and CD154, wherein said compound binds the CDprotein at the CD154-binding site. 20-24. (canceled)